Factors contributing to the success of a single-shot, multiyear PZP immunocontraceptive vaccine for white-tailed deer

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Human Wildlife Interactions Wildlife Damage Management, Internet Center for Spring 2009 Factors contributing to the success of a single-shot, multiyear PZP immunocontraceptive vaccine for white-tailed deer Lowell A. Miller USDA/APHIS/Wildlife Services National Wildlife Research Center, Fort Collins, CO, lowell.a.miller@aphis.usda.gov Kathleen A. Fagerstone USDA/APHIS/Wildlife Services National Wildlife Research Center, Fort Collins, CO Donald C. Wagner Pennsylvania State University, dwagner@psu.edu Gary J. Killian USDA/APHIS/Wildlife Services National Wildlife Research Center, Las Cruces, NM Follow this and additional works at: http://digitalcommons.unl.edu/hwi Part of the Environmental Health and Protection Commons Miller, Lowell A.; Fagerstone, Kathleen A.; Wagner, Donald C.; and Killian, Gary J., "Factors contributing to the success of a singleshot, multiyear PZP immunocontraceptive vaccine for white-tailed deer" (2009). Human Wildlife Interactions. 34. http://digitalcommons.unl.edu/hwi/34 This Article is brought to you for free and open access by the Wildlife Damage Management, Internet Center for at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Human Wildlife Interactions by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln.

Human Wildlife Confl icts 3(1):103 115, Spring 2009 Factors contributing to the success of a single-shot, multiyear PZP immunocontraceptive vaccine for white-tailed deer LOWELL A. MILLER, USDA/APHIS/Wildlife Services National Wildlife Research Center, 4101 LaPorte Avenue, Fort Collins, CO 80521, USA lowell.a.miller@aphis.usda.gov KATHLEEN A. FAGERSTONE, USDA/APHIS/ Wildlife Services National Wildlife Research Center, 4101 LaPorte Avenue, Fort Collins, CO 80521, USA DONALD C. WAGNER, 324 Hennning Building, Pennsylvania State University, University Park, PA 16802, USA GARY J. KILLIAN, USDA/APHIS/Wildlife Services National Wildlife Research Center, Las Cruces, NM 88005, USA Abstract: We evaluated 6 different porcine zona pellucida (PZP) preparations used as a single-shot vaccine for multiyear contraception of captive white-tailed deer (Odocoileus virginianus). The study compared 2 PZP preparation technologies from ImmunoVaccine Technologies (IVT) and National Wildlife Research Center (NWRC) over a 7-year period. The study compared both the use of oil in an emulsion and in suspension delivery, as well as replacement of the oil with an alum adjuvant. The study demonstrated that the oil emulsion adjuvant provided the longest lasting response. PZP isolated by the IVT provides a longerlasting response than the preparation used by NWRC. The SpayVac TM and IVT-PZP vaccines presented in an emulsion form with AdjuVac produce a single-shot immunocontraceptive vaccine lasting up to 7 years. Key words: human wildlife confl icts, immunocontraception, Odocoileus virginianus, porcine zona pellucida, single-shot PZP vaccine, white-tailed deer Immunocontraceptive vaccines prevent conception by stimulating production of antibodies that bind with and neutralize proteins or hormones essential for reproduction (Curtis et al. 2008, Miller et al. 2008). Porcine zona pellucida (PZP) vaccines prepared using zonae pellucida isolated from pig ovaries are one of the most studied immunocontraceptive vaccines in wildlife. The zona pellucida is a cellular glycoprotein layer surrounding the mammalian oocyte. During fertilization, sperm bind to receptors on the outer surface of the oocyte and penetrate the zona pellucida. Anti-PZP antibodies block sperm binding and penetration into the ovum and may also interfere with follicle maturation and ovulation (Dunbar 1989, Aitken et al 1996). Females treated with PZP vaccines typically experience multiple infertile estrous cycles throughout the breeding season (Killian and Miller 2000, Miller and Killian 2000, Miller et al. 2000). PZP vaccines have been used to contracept feral horses (Equus caballus; Kirpatrick et al. 1990, Kirpatrick et al. 1992), whitetailed deer (Odocoileus virginianus; Curtis et al. 2007, Killian et al. 2008, Rutberg and Naugle 2008), coyotes (Canis latrans; Miller et al. 2006), domestic sheep (Ovis aries; Stoops et al. 2006), and captive exotic species (Frank et al. 2005). Based on the encouraging results of early investigations, we began studies with the PZP vaccine in white-tailed deer during 1992. We hoped to develop a single-shot contraceptive vaccine that was safe for the animals and had multiyear efficacy. White-tailed deer were then, as they are now, of interest as a target species because of inadequate control methods available to deal with dense urban and suburban deer populations in the northeastern United States (Hussain et al. 2007, Bissonettee et al. 2008, DeNicola and Williams 2008, Mastro and Conover 2008, Ng et al. 2008). In an early study, we immunized white-tailed deer with a 1-ml injection containing 500-μg PZP in Freund s Complete Adjuvant (FCA) followed by multiple booster injections, each with 1-ml injection containing 300-μg PZP in Freund s Incomplete Adjuvant (FIA). In the first year, the prime dose was given in August and 2 boosts were given in ember and ober. In ember, the does were exposed to bucks. In the second year, does were given 1 or 2 boosts, depending on their current PZP titer.

104 Human Wildlife Confl icts 3(1) We continued the study for 4 more years without additional boosts. The does exhibited 100% contraception the first year, 89% over 3 years, and 76% over 6 years (Miller and Killian 2000). Given the multiple injections and the high dose of PZP required to induce infertility, we recognized that this approach was not practical for field applications. In a later field study, we showed that a prime and boost of 100 ug of PZP emulsified in FCA and FIA could provide multiyear contraception (Curtis et al. 2008). Few studies have reported that a single-injection vaccine can provide immunization with effectiveness up to 2 years (Fraker et al. 2002, Liu et al. 2005, Locke et al. 2007, Turner et al. 2007, Rutberg and Naugle 2008). In mice, a single injection with a vaccine containing influenza viral peptides and adjuvant (Adjumer ) in mice caused antibody titers to increase gradually up to 6 months, but titers began to decline at 9 months (Payne et al. 1995). Their study suggested that for infertility to be achieved with a single injection, the vaccination would need to be given months before the onset of reproductive activity (i.e., the rut period) so that an adequate titer level could develop for contraception to occur. Adjuvants are critical in vaccines for the development of high antibody titers in response to vaccination. Our work on a new adjuvant began in 1998 after learning that the U.S. Food and Drug Administration (FDA) would not approve the use of vaccines containing FCA because this adjuvant was associated with significant reactions at the site of injection. Consequently, we developed a new oil-based adjuvant called AdjuVac, containing <200 μg of killed Mycobacterium avium per dose. M. avium is a common nonpathogenic bacterium to which most wildlife and domestic animals worldwide have been exposed. The experiments described in the current paper represent the last phase of the study conducted with the PZP vaccine in white-tailed deer. The objectives of the study was to compare the effectiveness of 6 different single shot vaccine formulations, containing different PZP preparations and different adjuvants or delivery systems. Methods Deer Research Center at Pennsylvania State University White-tailed deer used in this study were born, raised, and maintained at the Pennsylvania State University (PSU) Deer Research Center. The original source of animals for the PSU deer herd was the free-roaming white-tailed deer population from central Pennsylvania. During our study, the PSU deer facility encompassed 54 ha of natural forest habitat and was divided into 9 outdoor paddocks ranging in size from 0.1 to 1.5 ha. Vegetation on the open areas consisted of a mixture of clover and orchard grasses, but most of the land was covered with dense eastern deciduous forest that had little understory vegetation. Deer were kept at a density of 25 to 37 animals per ha. During the nonbreeding season, treated female deer were isolated from males. To test vaccine contraceptive efficacy, 4 bucks of proven sire ability were confined with the does each year from the first week of ember through the end of the following ruary. PZP vaccine preparations The heat-soluble PZP preparation used for the PZP was prepared by Dr. Irwin Liu (University of California at Davis) following the method described by Dunbar and Raynor (1980). PZP was provided to NWRC in phosphate buffered saline (PBS ) buffer at a protein concentration of 1 mg/ml. It was used in either a 200-μg or 500- μg protein dose prepared as an emulsion with AdjuVac for administration in a 1-ml volume. This preparation will henceforth be referred to as NWRC-PZP. ImmunoVaccine Technologies (IVT) PZP vaccine preparations ImmunoVaccine Technologies (IVT) of Halifax, a Scotia, Canada, developed the IVT-PZP preparation using the Yurewicz et al. (1983) procedure to isolate zona. This method is similar to that described by Dunbar and Raynor (1980). The IVT procedure consisted of homogenizing the ovaries in Tris buffer by passage through a commercial meat grinder. The resulting oocytes were cleared from cellular debris by successive passages of the ground tissue through a series of nylon screens of decreasing pore size. The oocytes were then passed

PZP vaccine Miller et al. 105 through a homogenizer to release the zona pellucida. We emulsified this PZP with AdjuVac to create the IVT-PZP vaccine. We used SpayVac (an IVT-PZP preparation encapsulated in liposomes; Brown et al. 1997) in 3 other vaccine preparations. These were SpayVac emulsified with AdjuVac, lypohilized SpayVac suspended in AdjuVac, and SpayVac mixed with alum as the adjuvant. Experimental design We divided 30 deer into 6 treatment groups of 5 does each (Table 1). The first 4 treatment groups were vaccinated with variations of the IVT-PZP preparation. Group 1 was vaccinated with SpayVac (IVT-PZP encapsulated in liposomes) suspended in AdjuVac. Group 2 was vaccinated with IVT-PZP (without the liposomes) suspended in AdjuVac. Group 3 was vaccinated with lyophilized SpayVac suspended in Adju- Vac. Group 4 received SpayVac combined with alum. Lyophilized (freeze-dried) vaccine used in Group 3 evaluated whether a suspension would produce a long-lasting contraceptive effect like that seen with the emulsion in the standard vaccine preparations. The SpayVac in alum used in Group 4 was intended to evaluate the utility of alum suspension as an adjuvant for producing long-term contraception. All IVT-PZP-treated deer were vaccinated with 200 μg of PZP that contained approximately 35% protein. The IVT-PZP preparation contained some precipitate that became part of the vaccine preparation. Groups 5 and 6 were vaccinated with 200-μg and 500-μg doses, respectively, of NWRC-PZP as measured by Lowry assay. Vaccines were prepared by producing a 1:1 emulsion volume:volume with AdjuVac. We administered all PZP vaccine formulations in mid-y 2000. We took 10 ml of blood from does in all groups except for Group 2, which was vaccinated during y 2001. Fawning data were available for untreated does exposed to bucks in the Penn State herd during each year of study. While these does were not sham-treated, they were handled in the summer and fall, as were the treated does, when they received health vaccinations. We considered the fawning rates of the untreated does to represent normal fertility rates for does in the Penn State herd. Laboratory methods We collected 10 ml of blood by jugular venipuncture from the study deer in y, ember, ember, and ruary. We used the sample to determine antibody titers, progesterone levels, and pregnancy-specific protein B in samples collected in ruary. Anti-PZP titers for groups using IVT-PZP and the NWRC-PZP 200 μg/dose as the antigen were determined by ELISA at the IVT laboratories. This method reports the titer as a percentage of a standard rabbit anti-pzp titer, which is considered 100% (Brown et al. 1997). Anti-PZP titers for the NWRC-PZP 500 μg/ dose vaccine were measured, using ELISA at the NWRC laboratories. Titers were calculated as the last serial dilution in which the treated sample was at least twice the absorbance of the pretreated sample of the same animal. In the NWRC laboratory method to determine anti- Table 1. Experimental design defining the nature of the vaccine treatments and adjuvants. Groups 1 4 represent PZP produced by IVT mixed with different adjuvant preparations. Groups 4 and 5 represent 200 and 500 μg of PZP from NWRC, both made into an emulsion. Group Vaccine Treatment Liposomes Adjuvant Adjuvant-Vaccine Mixture 1 IVT-PZP(200 μg) SpayVac Yes AdjuVac Emulsion 2 IVT-PZP(200 μg) No AdjuVac Emulsion 3 IVT-PZP(200 μg) SpayVaclyophilized Yes AdjuVac Suspension 4 IVT-PZP(200 μg) SpayVac Yes Alum Suspension 5 NWRC-PZP(200 μg) No AdjuVac Emulsion 6 NWRC-PZP(500 μg) No AdjuVac Emulsion

106 Human Wildlife Confl icts 3(1) body titers, 100 ng of PZP antigen was placed in each well of a micro-titer plate. Sea Block TM (Pierce Chemical, Rockford, Ill.) was used to prevent binding of antibodies to the plastic in the ELISA plate. Deer serum was serially diluted from 1:1,000 to 1:128,000 in PBS containing Sea Block. Antibodies in the deer serum to the native PZP antigen on the plate were directed with the following linkages: deer anti-pzp binds to PZP on the plate, rabbit anti-deer IgG binds to the deer IgG, goat anti-rabbit-peroxidase binds to the rabbit IgG. Tetramethylbenzidine was used to develop the color, and 2M H 2 SO 4 was used to stop the reaction. The color intensity of the sample was read at 450 nm with a Dynatech MR 5000 ELISA plate reader. Plasma progesterone levels were assayed by the coat-a-tube RIA method (Diagnostic Products, Los Angeles, Calif.), following the manufacturer s recommended procedure. Assays for Pregnancy-Specific Protein B (PSPB) were performed by ELISA on samples submitted to BioTracking LLC (Moscow, Ida.). Behavioral observations We monitored estrous cycles in deer to determine the frequency of estrus and to learn about the mechanism of multiyear contraception. To determine when the does were in estrus, trained observers monitored the behavior of bucks toward the treated does. Three observation periods of >30 minutes each were scheduled daily from ember 7 through ruary 12, followed by 2 periods daily until ruary 28. Behavioral activity by the buck was recorded on an observation sheet for a range of activities, including sniffing genitalia, pursuit of the female, aggressive guarding, and copulation (Killian and Miller 2000). Ultrasonography and fawning data We performed trans-rectal ultrasonography in late uary or early ruary of each year to determine which does were in the first trimester of gestation. Blood drawn on the same day was tested for progesterone concentration and PSPB. From May through ember, does were observed daily for evidence of fawning. We recorded fawning dates and the number of fawns born and compared them to observations on behavioral estrus to estimate the date of conception. Definitions of contraception and fertility Does that were exposed to bucks and showed signs of estrus, but did not fawn were considered contracepted or infertile. Does in each group were monitored for contraceptive longevity and remained on the study until they had twins. Does >2 years old and having 1 fawn were assumed to be sub-fertile because this is less than the average fawning rate of 2 fawns in untreated does in the Penn State herd. Results Contraception The average fawning rate of the Penn State herd for 84 untreated does from 2001 through 2007 was 1.8 fawns per doe, with an average doe age of 4 years (Table 2). Number of fawns born varied with does age. For 2-year-olds, the Table 2. Fawning data for untreated does in the Penn State University deer herd during the same years that the contraceptive trial was conducted. Year % infertile n Average doe age % does with 1 fawn % does with 2 or more fawns 1 0 9 4.0 67 33 1.4 2 0 26 3.1 23 77 1.8 3 0 8 4.7 13 87 2.0 4 0 10 4.2 20 80 2.0 5 0 13 4.2 0 100 2.0 6 0 8 4.6 13 87 1.9 7 0 10 4.5 20 80 1.8 Average fawns/doe

PZP vaccine Miller et al. 107 average number of fawns born was 1.7. For does >2 years old, the average number of fawns born was 2.0. Groups 1 and 2, which combined SpayVac or IVT-PZP into an emulsion with AdjuVac, had the longest contraceptive effect, with 80% of the deer contracepted for 5 to 7 years (Table 3). Unfortunately, Group 1 was terminated after 5 years because of lack of funding, with 80% of the treated deer still contracepted. One doe in Group 2 had twins in year three and was dropped from the study. In year five, 1 of the 4 remaining does in this group had a single fawn, reducing the rate of contraception to 60%. However, because the birth of a single fawn did not represent a complete breakthrough from immunological contraception, all 4 does were kept for another year. In years six and seven, the doe that had a fawn in year five did not fawn, restoring the contraception rate to 80% (Table 2). Does in Group 3 were all contracepted in the first year, one of the 4 does conceived the second year, two of the 4 does had fawns during the third year, and all fawned during the fourth year (Table 3). Consequently, this group was dropped from the study at the end of year four. Four of the 5 does in Group 4 each had 1 fawn in the first year (Table 3). Consequently, this group was removed from the study after the first year. Groups 5 and 6 received 200 μg and 500 μg of NWRC-PZP, respectively, in an emulsion made with AdjuVac. For Group 5, 1 doe had 2 fawns the first year, for an 80% contraception rate (Table 3). However, because all 5 does produced 2 fawns in the second year, Group 5 (200 μg of NWRC-PZP) was subsequently removed from the study. Does receiving 500 μg of PZP were 100% contracepted the first year, but 4 of 5 does produced fawns the second year (Table 3). This 20% contraception rate remained until year five, when the remaining doe produced 2 fawns. The number of fawns that could be produced by the different groups, had they not be contracepted, was estimated from the average fawning rates of untreated does in the herd during the same years. This estimate of fecundity for the 5 years when Group 1 was on the study was 46 fawns compared to 64 fawns for 7 years of study with Group 2 (Table 3). Considering that actual numbers of fawns produced for these 2 groups during the periods of study were 2 and 4 respectively, fecundity was reduced greater than 90% by the vaccines containing IVT-PZP. Antibody titers Comparing the anti-pzp titers of all of the groups, Group 1 (SpayVac; Figure 1a) had the greatest initial immune response of all groups, with a peak antibody titer at 18 months and a titer of 80 to 100% of the rabbit standard during the first 2 years. Although the titer began to drop after 18 months, it was sustained at 40% to the end of the 5-year study. The rise in antibody titer for Group 2 IVT-PZP (Figure 1b) was slower than that observed for Group 1, but by year three, both groups had similar titers. The antibody response patterns for Group 2, and to a lesser extent for Groups 1 and 3, included a self-boosting of titers, which was evident as titers increased without additional antigen injections (Figures 1a and 1b). Notably, for Groups 1 and 2, the high rates of contraception were maintained even with lower titers in years four through seven. Deer in Group 3 that were given freeze-dried SpayVac suspended in AdjuVac, were 100% contracepted in the first year, but contraception rates dropped off to 75%, 50%, and 0% in years two, three, and four, respectively (Table 3). Titers for Group 3 were high through year two, but dropped in years three and four (Figure 1c), indicating that freeze-dried SpayVac in suspension was less effective for long-term contraception than the non-freeze-dried emulsion form of SpayVac. SpayVac with alum adjuvant produced a large variation in the immune responses, and only 1 doe produced a good immune response (Figure 2a). Titers for Group 5, (200-μg NWRC-PZP mixed with AdjuVac) were relatively high in the first year (Figure 2a), with an 80% contraception rate. However, average titers dropped dramatically in year two, and all of the does conceived. Antibody titers determined for Group 6, treated with (500-μg NWRC-PZP mixed with Adju- Vac ) were very high immediately after injection, but declined over the next 2 years (Figure 2b). In contrast to the IVT-PZP used in Groups 1 and 2 (Figures 1a and 1b), the NWRC-PZPtreated does in Groups 5 and 6 did not appear

108 Human Wildlife Confl icts 3(1) Table 3. Number of contracepted female deer that gave birth and number of fawns produced by them. All does were given a single vaccine dose of 200 μg on y 10, 2000, except Group 3 does, which were immunized in y 2001. Fawning Year Group 1 SpayVac-AdjuVac n = 5 Group 2 IVT-PZP-AdjuVac n = 5 IVT PZP preparations NWRC preparations Group 3 Lyophilized SpayVac-AdjuVac n = 4 Group 4 SpayVac-Alum n = 5 Group 5 NWRC-PZP (200 μg)-adjuvac n = 5 Group 6 NWRC-PZP (500 μg)-adjuvac n = 5 Females Fawns Females Fawns Females Fawns Females Fawns Females Fawns Females Fawns Year 1 0 0 0 0 0 0 4 4 1 2 0 0 Year 2 0 0 1 0 0 0 5 10 4 6 Year 3 0 0 1 1 2 2 4 2 Year 4 1 1 1 2 4 2 Year 5 1 1 1 1 3 6 5 2 Year 6 1 1 Year 7 1 1 Total fawns % Reduction in fecundity 1 2 6 8 4 12 12 96 94 75 43 25 74 1 Fecundity = expected rate of fawn production for the number of does in the treatment group for the duration of the study, using the average fawning rates of untreated does (Table 2) for the same years. Percentage reduction in fecundity was determined by dividing the difference between the expected fawning rate and the actual fawning rate by the expected fawning rate.

PZP vaccine Miller et al. 109 % of reference 120 100 80 60 40 Figure 1a. Group 1: Ab titer (PZP Lipo06sp). SpayVac as an emulsion with AdjuVac. 20 0 % of reference 90 80 70 60 50 40 30 20 10 0 Figure 1b. Group 2: Ab titer (PXPb200spwpd). IVT-PZP as an emulsion with Adju- Vac. 60 50 Figure 1c. Group 3: SpayVac, freeze-dried, as a suspension in AdjuVac. 40 % of reference 30 20 10 0 70 60 Figure 1d. Group 4: SpayVac absorbed onto alum. % of reference 50 40 30 20 10 0 Figure 1, a d. Each group represents a different PZP with different adjuvant preparations and delivery methods. Antibody titers are presented as a percentage of a 100% anti-pzp rabbit standard. y 2000 to ruary 2006 represents the month-years post treatment throughout the study. The graph ends at the point that all deer in the group became pregnant and the group was dropped from the study. Each data point represents the mean ± SE.

110 Human Wildlife Confl icts 3(1) % of reference 120 100 80 60 40 20 0 Figure 2a. Group 5: 200 μg of NWRC- PZP presented as an emulsion with AdjuVac, antibody titer presented as a percentage of a rabbit standard as 100%. Antibody Titer (x 1000) 250 200 150 100 50 Figure 2b. Group 6: 500 μg of NWRC- PZP presented as an emulsion with AdjuVac antibody titer presented as a 1/1000 1/256,000 serial dilution end point. 0 Figure 2 a b. Groups 5 and 6 represent 2 concentrations of the NWRC-PZP vaccine serial dilution end point. y 2000 is the date of the vaccination, with y 2000 to ruary 2006 as the months and years of post-treatment throughout the study. The graph ends at the point that all deer in the group became pregnant, and the group was dropped from the study. Each data point represents the mean (±SE). to stimulate self-boosting, and the decreasing of antibody titers (Figures 2a and 2b) resulted in greater pregnancy rates in subsequent years (Table 3). Behavioral observations PZP-treated deer had multiple estrous cycles every year of the 5-year study, with the multicycling ceasing only when they became pregnant. The number of estrus events for all PZPtreated deer ranged from 1.5 to 3.0 per season, with an average breeding season length of 150 days. This compares to 0.2 to 0.5 estrus events for does in the untreated herd and an average breeding period of 42 days per season. The 4 does in IVT-PZP Group 2 averaged 1.75 estrus events in year seven of the trial, demonstrating that cycling was still occurring, although none of the does had fawns. Progesterone and pregnancyspecific protein B determinations Blood samples taken from deer anually in y, ember, ember, and ruary were assayed for serum progesterone concentrations. Despite the fact that the PZP-treated deer showed signs of estrus and breeding behaviour during ember and December, the only consistent elevation in serum progesterone observed was in the ruary blood samples of SpayVac Group 1, IVT-PZP Group 2, and NWRC-PZP 500-μg Group 6 (Figures 3a, 3b and 3c). This consistent increase in serum progesterone in ruary in the PZP-treated does was not associated with pregnancy. Blood serum was taken from 6 adult, proven-breeder does that were not bred during the 2006 2007 breeding season. Because these unbred, untreated does showed a similar elevation of serum progesterone in ruary (5.6 ± 0.4 SE), we concluded that this elevation of progesterone may be normal for cycling, nonpregnant does as they return to an anestrous state in the nonbreeding season. In contrast to serum progesterone concentrations, PSPB levels with a mean of 0.3 (non-

PZP vaccine Miller et al. 111 Progesterone (ng/ml) 8.00 7.00 6.00 5.00 4.00 3.00 2.00 Figure 3a. Group1: Progesterone data of SpayVac presented as μg/ml. 1.00 0.00 Progesterone (ng/ml) 6 5 4 3 2 1 Figure 3b. Group 2: Progesterone data IVT-PZP presented as ng/ml. 0 Progesterone (ng/ml) 8.00 7.00 6.00 5.00 4.00 3.00 2.00 Figure 3c. Group 6: Progesterone data of NWRC 500 μg presented as ng/ml. 1.00 0.00 Figure 3 a c. Progesterone peaks present in the uary ruary bleed in the SpayVac group (3a), the IVT group (3b), and the 500-μg NWRC group (3c). y 2000 to ruary 2006 represents the month and years of post-treatment throughout the study. Each data point represents the mean (±SE). pregnant values >1.0) were well-correlated with ultrasound and fawning results. The PSPB levels were correlated with fawning (r = 0.83, P <0.0001). Injection-site reactions There were no injection-site reactions in any of the treatment groups observed throughout the study.

112 Human Wildlife Confl icts 3(1) Discussion This is the first study of a single-injection PZP vaccine for white-tailed deer that results in contraception rates of 80% for 5 to 7 years. Based on the performances of the different vaccine preparations, it is clear that the long-lasting contraceptive effect is related to the vaccine design and the adjuvant used. The longest contraceptive effect was achieved in Groups 1 and 2 that included PZP produced by IVT and was made into an emulsion with the NWRC adjuvant AdjuVac. Both of these groups showed periodic boosting of the antibody titer, which may be central to the long-lasting contraceptive effect. Because the PZP-contracepted does continued to cycle throughout the 7-year study, we conclude that successful PZP immunocontraception depends on long-lasting, high titers of high-affinity antibodies and is unlikely the result of PZP-immune complexes resulting in ovarian damage. The immune system is unable to maintain the high antibody responses for long periods in the absence of a stimulating antigen. Therefore, for a successful immunocontraceptive response, the antigen must be retained in the immune system for months or years (Burton et al. 1994). The follicular dendritic cells in the lymph node that drains the site of injection may provide an answer for the long-term immune response found with some antigens. To induce immunocontraception that lasts months to years, there must be a continued stimulation of these B-cells by the injected antigen (Burton et al. 1994). Antigen preparation Differences between the methods used to harvest oocytes for the IVT and NWRC antigen preparations may have affected the length of contraceptive response. For the NWRC preparation, the oocytes in the ovaries were cleanly released by 2 beds of 500 razor blades, which enabled the rapid isolation of oocytes with minimal disruption of other ovarian tissue. In contrast, for the IVT preparation, the ovaries were processed in a meat grinder as the initial step for isolating the oocytes. Although most of the remaining steps were similar, the initial material in the IVT preparation probably contained more ovarian tissue. In contrast to the long-term efficacy achieved with the IVT-PZP groups, the NWRC-PZP preparations performed well only for 1 to 2 years. Adjuvants The oil-based adjuvant AdjuVac was an important component of the single-injection vaccine, contributing to both the antibody titer produced and the long-term contraceptive response. The benefit of AdjuVac was clearly evident when we compared it to the poor response when alum was used as the adjuvant. The liposomes in SpayVac increased the longterm contraceptive effect in other studies and species. In this study, the IVT-PZP performed almost as well as the SpayVac, although it is possible that the addition of AdjuVac diminished the need for the liposomes in SpayVac. Presumably, the liposomes facilitated a significant immune response when the vaccine was combined with a less-effective adjuvant. Multi-year contraceptive effectiveness has occurred in gray seals (Halichoerus grypus; Brown et al. 1997), fallow deer (Dama dama; Fraker et al. 2002), and white-tailed deer (Odocoileus virginianus; Locke et al. 2007) as a result of a single administration of SpayVac using Freund s Complete Adjuvant. For horses, Liu et al. (2005) demonstrated a multiyear contraceptive effect when PZP was injected with Freund s adjuvant, and Turner et al. (2007) also was able to achieve a multiyear contraceptive effect with PZP in a slow-release microbead. However, in the Liu and Turner studies, the contraceptive effect was limited to 2 years, as we observed with the NWRC-PZP prepared by Liu, which is considerably less than the 5 7 years achieved with the IVT-PZP in this study. Emulsion This study showed that an adjuvant prepared with the antigen in the form of an emulsion provides longer lasting contraception than that observed with a suspension of freeze-dried PZP. The antigen administered in a single dose must be retained in the body long enough to produce specific antibodies that will bind with the antigen to form immune complexes (Bachmann et al. 1996). It appears that a stiff emulsion (similar to mayonnaise) is important for the retention of the intramuscular dose of vaccine.

PZP vaccine Miller et al. 113 The seasonal breeder: endogenous PZP antigen results in self-boosting It is possible that the success of the PZP-IVT vaccines is affected by the fact that deer are a seasonal multicycling animal. Each season when does enter a breeding condition, it is likely that the zona pellucida antigen is presented to the immune system, resulting in a self-boosting effect. It is possible the particulate nature of the IVT-PZP preparations resulted in a longerboosting effect and contributed to the longer contraceptive effect. There does not appear to be any long-term negative health effect from the PZP contraception (Miller et al. 2001). Immunocontraception holds the promise to help resolve deer human conflicts (Bingham 2007). The possibility of a single-injection PZP contraceptive that would be effective as a contraceptive for multiple years increases the usefulness of an injectable contraceptive for the reproductive control of wildlife. Literature cited Aitken, R. J., M. Patterson, and M. van Duin. 1996. The potential of the zona pellucida as a target for immunocontraception. American Journal of Reproductive Immunology 35:175 180. Bachmann. M. F., B. Odermatt, H. Hengartner, and R. M. Zinkernagel. 1996. Induction of long-lived germinal centers associated with persisting antigen after viral infection. Journal of Experimental Medicine 183:2259 2269. Bingham, E. 2007. Birth control is not for everyone (or everything). Human Wildlife Confl icts 1:12. Bissonette, J. A., C. A. Kassar, and L. J. Cook. 2008. Assessment of costs associated with deer vehicle collisions: human death and injury, vehicle damage, and deer loss. Human Wildlife Confl icts 2:17 27. Brown, R. G., W. D. Bowen, J. D. Eddington, W. C. Kimmins, M. Mezei, J. L. Parson, and B. Pohajdak. 1997. Evidence for a long-lasting single administration vaccine in wild grey seals. Journal of Reproductive Immunology 35:43 51. Burton, G. F., A. K. Szakal, Z. F. Kapasi, and J. G. Tew. 1994. The generation and maintenance of antibody and B cell memory: the role of retained antigen and follicular dendritic cells. Pages 35 50 in G. L. Ada, editor. Strategies in vaccine design. Landes, Austin, Texas, USA. Curtis, P. D., M. E. Richmond, L. A. Miller, and F. W. Quimby. 2007. Pathophysiology of whitetailed deer vaccinated with porcine zona pellucida immunocontraceptive. Vaccine 25:4623 4630. Curtis, P. D., M. E. Richmond, L. A. Miller, and F. W. Quimby. 2008. Physiological effects of gonadotropin-releasing hormone immunocontraception on white-tailed deer. Human Wildlife Confl icts 2:68 79. DeNicola, A. J., and S. C. Williams. 2008. Sharpshooting suburban white-tailed deer reduces deer vehicle collisions. Human Wildlife Confl icts 2:28 33. Dunbar, B. S., 1989. Ovarian antigens and infertility. American Journal of Reproductive Immunology 21:28 31. Dunbar, B. S., and B. D. Raynor. 1980. Characterization of porcine zona pellucida antigens. Biology of Reproduction 22:941 954. Fraker, M. A., R. G. Brown, G. E. Gaunt, J. A. Kerr, and B. Pohajdak. 2002. Long-lasting singledose immunocontraception of feral fallow deer in British Columbia. Journal of Wildlife Management 66:1141 1147. Frank, K. M, R. O. Lyda, and J. F. Kirkpatrick. 2005. Immunocontraception of captive exotic species: IV. Species differences in response to the porcine zona pellucida vaccine, timing of booster inoculations, and procedural failures. Zoo Biology 24:349 358. Hussain, A., J. B. Armstrong, D. B. Brown, and J. Hogland. 2007. Land-use pattern, urbanization, and deer vehicle collisions in Alabama. Human Wildlife Confl icts 1:89 96. Killian, G. J., and L. A. Miller. 2000. Behavioral observation and physiological implications for white-tailed deer treated with two different immunocontraceptives. Eastern Wildlife Damage Management Conference 9:283 291. Killian, G. J., J. D. Thain, N. K. Diehl, J. C. Rhran, and L. A. Miller. 2008. Four-year contraceptive rates of mares treated with single-injection porcine zona pellucide and GnRH vaccine and intrauterine devices. Wildlife Research 35:531 539. Kirkpatrick, J. F., I. K. M. Liu, and J. W. Turner, 1990. Remotely-delivered immunocontraception in feral horses. Wildlife Society Bulletin 18:326 330. Kirkpatrick, J. F., I. K. M. Liu, J. W. Turner, R. Naugle, and R. Keiper, 1992. Long-term effects of

114 Human Wildlife Confl icts 3(1) porcine zonae pellucidae immunocontraception on ovarian function in feral horses (Equus caballus). Journal of Reproductive Fertility 94: 437 444. Liu, I. K. M., J. W. Turner, E. M. G. Van Leeuwen, D. R. Flanagan, J. L. Hedrick, and K. Murata. 2005. Persistence of anti-zonae pellucidae antibodies following a single inoculation of porcine zonae pellucidae in the domestic equine. Reproduction 129:181 190. Locke, S. L., M. W. Cook, L. A. Harveson, D. S. Davis, R. R. Lopez, N. J. Silvy, and M. A. Fraker. 2007. Effectiveness of SpayVac for reducing white-tailed deer fertility. Journal of Wildlife Disease 43:726 730. Mastro, L. L., M. R. Conover, and S. N. Frey. 2008. Deer vehicle collision prevention techniques. Human Wildlife Confl icts 2:80 92. Miller, L. A. 2002. In search for the active PZP epitope in white-tailed deer immunocontraceptive. Vaccine 20:2735 2742. Miller, L. A., K. Bynum, and D. Zemlicka. 2006. PZP immunocontraception in coyote: a multiyear study with three vaccine formulations. Vertebrate Pest Conference 22:88 95. Miller, L., A., K. Crane, S. Gaddis, and G. J. Killian. 2001. PZP immunocontraception: long-term health effects on white-tailed deer. Journal of Wildlife Management 65:941 945. Miller, L. A., J. P. Gionfriddo, J. C. Rhyan, and K. A. Fagerstone, D. C. Wagner, and G. J. Killian. 2008. GnRH immunocontraception of male and female white-tailed deer fawns. Human Wildlife Confl icts 2:93 101. Miller, L. A., B. E. Johns, and G. J. Killian. 1999. Long-term effects of PZP immunization on reproduction in white-tailed deer. Vaccine 18:568 574. Miller, L. A., B. E. Johns, and G. J. Killian. 2000. Immunocontraception of white-tailed deer using native and recombinant zona pellucida vaccines. Animal Reproductive Science 63:187 195. Miller, L. A., and G. J. Killian. 2000. Seven years of white-tailed immunocontraception research at Penn State University: a comparison of two vaccines. Eastern Wildlife Damage Management Conference 9:60 69. Ng, J. W., C. Nielsen, and C. C. St. Clair. 2008. Landscape and traffi c factors infl uencing deer vehicle collisions in an urban environment. Human Wildlife Confl icts 2:34 47. Payne, L. G., S. Jenkins, A. Andrianov, and A. E. Roberts. 1995. Water-soluble phosphazene polymers for parenteral and mucosal vaccine delivery. Pages 473 493 in M. F. Powell and M. J. Newman, editors. Vaccine design: the subunit and adjuvant approach. Plenum, New York, New York, USA. Rutberg, A. T., and R. E. Naugle. 2008. Deer vehicle collision trends at a suburban immunocontraception site. Human Wildlife Confl icts 2:60 67. Stoops, M. A., I. K. Liu, S. E. Shideler, B. L. Lasley, R. A. Fayrer-Hosken, and K. Berirschke. 2006. Effect of porcine zonae pellucidae immunization on ovarian follicular development and endocrine function in domestic ewes (Ovis aries). Reproductive Fertility Development 18:667 676. Turner, J., W., I. K. M. Liu, D. R. Flanagan, A. T. Rutberg, and J. F. Kirkpatrick. 2007. Immunocontraception in wild horses: one inoculation provides two years of infertility. Journal of Wildlife Management 72:662 667. Turner, J. W., I. K. M. Liu, and J. F. Kirkpatrick. 1992. Remotely delivered immunocontraception in captive white-tailed deer. Journal of Wildlife Management 56:154 57. Yurewicz, E. C., A. G. Sacco, and M. G. Subramanian. 1983. Isolation and preliminary characterization of a purifi ed pig zona antigen (PPZA) from porcine oocytes. Biology of Reproduction 29:511 523.

PZP vaccine Miller et al. 115 LOWELL A. MILLER received his Ph.D. degree in Physiology and Immunology at Colorado State University, Fort Collins, Colorado. He is currently project leader for Reproductive Control Methods Project at the National Wildlife Research Center in Fort Collins, Colorado. The project is researching ways to induce infertility in overabundant species of wildlife. The project has developed a single injection GnRH contraceptive vaccine (GonaCon TM ) that has successfully contracepted the white-tailed deer, as well many other overabundant mammalian species. KATHLEEN A. FAGERSTONE is the manager of the Invasive Species and Technology Development Research Program at the USDA s National Wildlife Research Center in Fort Collins, Colorado. She obtained her B.S. degree in zoology from Colorado State University and her M.S. and Ph.D. degrees from the University of Colorado Boulder. In her current position, she oversees research projects to develop methods, including wildlife contraceptives, for dealing with problems caused by overabundant wildlife and invasive species. DONALD C. WAGNER obtained his B.S. degree in wildlife and fi sheries science at Pennsylvania State University in 1997. Since 2000, he has managed the Penn State Deer Research Center, where he has been involved with immunocontraceptive studies at the facility since 1996. GARY J. KILLIAN received his Ph.D. degree in reproductive physiology from Pennsylvania State University. Until 2006, he was a distinguished university professor at Penn State, where he collaborated with scientists at the National Wildlife Research Center, evaluating and testing immunocontraceptive vaccines in white-tailed deer, domestic and feral swine, and wild horses since 1991. Recently, he left Penn State to focus exclusively on collaborations with Lowell Miller and colleagues at the National Wildlife Research Center. His interests are fertility and disease management in overabundant wildlife and feral species. He is currently a reproductive physiologist with USDA/APHIS/WS and resides in New Mexico.